Caulobacter crescentus has a well defined life cycle in which a stalked cell divides repeatedly and asymmetrically to produce the stalked cell plus a new swarmer cell, which carries a constellation of surface structures, including one flagellum, pili, and a holdfast; all of these structures are assembled at the stalk distal pole at discrete times in the cell cycle. Flagellin and hook protein synthesis are regulated at the transcriptional level, and one of our goals is to understand how gene transcription is temporally regulated to give the observed patterns of periodic expression in the cell cycle. The nucleotide sequence and gene organization of the polycistronic hook operon will be determined and the polypeptides coded by it will be identified. S1 mapping of the transcriptional starts in this and the adjacent transcriptional units in the hook gene cluster will be carried out to locate regulatory sequence and to provide a direct assay for gene expression. A systematic mutational analysis of the sequences will be carried out to identify the promoters and the 5' regulatory regions that are responsible for periodic control of the hook operon. Proposals for a genetic and biochemical analysis of the trans-acting gene products that are required for hook and flagellin gene expression are also presented. To examine the mechanism of spatial localization in the cell envelope, the contruction of a series of fusion vectors for use in C. crescentus cells is planned. Polar localization of proteins will be examined by making in vitro gene fusions to the C-terminal portion of probe genes like LacZ and PhoA. This type of experiment will indicate whether the information for targeting polar proteins is contained within the protein sequence. Immunological approaches will be used to identify specialized membrane proteins at the cell pole.
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